CUT Domains as Therapeutic Targets. AmorChem KNOCK OUT 2017

Transcription

1 CUT Domains as Therapeutic Targets AmorChem KNOCK OUT 2017

2 Alain Nepveu McGill University Professor, Goodman Cancer Research Centre and Departments of Oncology, Biochemistry and Medicine. 1984: Ph.D : PDF, SUNY Stony Brook : PI, Ludwig Institute for Cancer Research 1992-present: McGill University Expertise in base excision repair, regulation of transcription, cell cycle progression. Funded by CIHR, NSERC, CRS, CCS. 2

3 Validation of CUX1 as a Therapeutic Target CUX1 and KRAS were shown to cooperate in the formation of mammary and lung tumors in mice. ~50% of mammary tumors in MMTV-CUX1 transgenic mice harbour a spontaneous mutation in Kras. lentiviral infections in mouse lungs show that CUX1 cooperates with Kras in lung tumor formation. CUX1 knockdown is synthetic lethal in human cancer cells harboring a RAS oncogene (KRAS, HRAS, NRAS, BRAF). CUX1 knockdown sensitizes cancer cells to radiotherapy. In contrast, CUX1 overexpression confers resistance. CUT domains of CUX1 confer resistance to radiation and to combined treatment with radiation and temozolomide in glioblastoma cells. 3

4 Activation of the RAS pathway leads to heightened production of reactive oxygen species CUT Domains Stimulate DNA Repair CUX1 C1 C2 C3 HD The CUT domains within the CUX1 protein stimulate two enzymes of the base excision repair (BER) pathway that repair oxidative DNA lesions: 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1). CUT domains are sufficient for recruitment to DNA damage and to confer resistance to ionizing radiation, temozolomide and combined treatment with radiation and temozolomide Activation of the RAS pathway leads to heightened production of reactive oxygen species (ROS), which causes oxidative DNA damage and ultimately, senescence. Cancer cells most often adapt to elevated ROS by increasing their capacity to repair oxidative DNA damage, through increased expression of CUX1. 4

5 The Opportunity I RAS-driven tumors represent ~30% of human cancers with NO targeted treatment for all types. Pancreas (59%), Small & large intestine (55%), Biliary tract (32%), Skin (27%), Ovary (19%), Lung (18%), Urinary tract (18%), Salivary gland (18%), Prostate (16%), Haematopoietic (15%), Upper aerodigestive tract (15%). RAS-driven tumor cells are acutely dependent on efficient oxidative DNA damage repair. This Achilles heel needs to be fully exploited for the development of new therapeutic strategies. Many drugs that inhibit base excision repair (BER: PARP1, APE1, DNA pol β enzymes are currently tested in the clinic with various treatment modalities. Major drawbacks of such approaches are: BER enzymes are essential to normal cell viability, since over 30,000 base alterations per day are produced endogenously in a normal human cell. Consequently, although inhibitors of BER enzymes increase cell killing within the tumor, they also cause severe adverse effects that considerably reduce the therapeutic window. CUX1 is not an essential gene synthetic lethality screens in human cells. Cux1 knockout mice are viable. CUX1 is needed only in situation of oxidative stress, as that caused by altered metabolism in RASdriven cancer cells or by ionizing radiation during radiotherapy. CUX1 inhibitors would thus have fewer side effects compared to that often seen with existing chemotherapies. 5

6 The Opportunity II CUX1 knockdown sensitizes cancer cells to ionizing radiation. In turn, ectopic expression of CUT domains (devoid of transcriptional potential) confers resistance to ionizing radiation. Chemotherapeutic agents used for radiosensitization cause adverse effects to normal cells (antimetabolites, platinums, temozolomide, microtubule inhibitors). In combination therapy, CUT domain inhibitors would sensitize cancer cells to radiotherapy, without causing adverse effects elsewhere in the body. Our expertise in the stimulation of DNA repair enzymes by auxiliary factors in cancer cells puts us in a strong and unique position for the development of CUT domain inhibitors. Dr. Zubaidah Ramdzan, who as a postdoctoral fellow discovered the role of CUT domains in DNA repair, is now a research associate in the laboratory and is committed to bring this project to completion. 6

7 Current State I We developed a high-throughput screening (HTS) assay to identify compounds that inhibit the DNA repair function of CUT domains. The assay uses a probe that contains an 8-oxoG base and a TAMRA fluorophore that resides next to a Black Hole Quencher-2 (BHQ-2) on the complementary strand. The glycosylase and AP/lyase of OGG1 creates a single-strand break which causes the release of a short oligo with the TAMRA fluorophore. The assay measures the increase in fluorophore emission. The screening assay has the properties required for screening compound libraries. The stimulation of OGG1 by CUT domains is on average 2.3, with a Z = The HTS assay is a validated assay. A first screen was performed at the Institut de Recherche en Immunologie et Cancérologie (IRIC) using 9583 compounds from 5 different libraries (Chembridge Diverset, Maybridge Hitfinder, Maybridge Selected, SPECS Selected, Cyclenium). We obtained a hit rate of 0.14%. Validation assays confirmed the specific activity of compounds. 7

8 Current State II Need to identify chemical libraries to screen Broad screen Focus libraries Optimization of protein stability would allow for more efficient screening. 8

9 Maturation Plan/Use of proceeds I 1. Our aim is to perform a high-throughput screen to identify small molecules that inhibit the stimulation of OGG1 by CUT domains. A go/no go approach will be adopted at each of the steps in order to identify a potential lead compound with characteristics allowing its further development as a CUT domain inhibitor for RAS-driven cancers. 2. Potential inhibitors identified in the screen will be validated in a second screen and by performing in vitro DNA repair assays with purified proteins, different fluorophore-based probes and radioactively labeled probes (Dr. Nepveu Lab). 3. Validated hits will first be clustered based on their common structural features. Chemotype series having more than one active compounds will be prioritized. A first round of structure-activity relationship (SAR) studies will be conducted. Hit derivatives will be tested in HTS and in vitro DNA repair assays. Medicinal chemistry could be performed under the supervision of Dr. Anne Marinier, at IRIC. NuChem Therapeutics could also undertake this work. 4. Structural analysis of CUT domains will be carried out by X-ray crystallography in the laboratory of Dr. Bushan Nagar, Dep. of Biochemistry, McGill. Interactions of compounds with CUT domains will be investigated using isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and, when compounds emit fluorescence, by fluorescence polarization assays. Structural information will serve in the rational design of modifications in the next rounds of synthesis. 9

10 Maturation Plan/Use of proceeds II 5. In vitro pharmacokinetic profiling of hit compounds will be initiated once a trackable SAR has been obtained with the lead series and acceptable activity has been demonstrated by a few analogs. The most promising molecules will be selected for PK assessment in vivo in the rat. PK studies will be conducted at the Biopharmacy Platform of Université de Montréal. 6. Potential Lead Compounds will be assessed for their toxicity and their capacity to reduce the viability of RAS-driven cancer cells and to sensitize cancer cells to various doses of ionizing radiation using clonogenic and proliferation assays. Their effect on DNA repair in cells will be measured using comet assays at various ph and immunostaining for γ-h2ax DNA repair foci.(dr. Nepveu Lab). 7. In vivo proof of concept will be done in several mouse models (Dr. Dankort, McGill U.) and patient-derived tumors maintained as xenografts (Drs. Morag Park, Peter Siegel, McGill U.). Radiotherapy of tumor xenografts will be performed using the X- RAD SmART, a small animal image-guided irradiation system equipped with high resolution CT scan for tumor guidance with integrated bioluminescence imaging platform, under the supervision of Dr. A. Nepveu and Dr. J. Seuntjens, medical physicist at the McGill University Health Center. 10

11 Maturation Plan/Use of proceeds III The budget for each of these steps is as follows: High-Throughput Screening and Validation : $194,250 Research Associate, 1 FTE for 1 year; Screening at IRIC; Oligonucleotides, OGG1 enzyme, Validation assays (TAMRA-, FAM-based probes, radioactive probes; Screening of hit derivatives. Medicinal Chemistry : $180,000 Research Assistant, 1 FTE for 1 year; Chemical reagents; Solvents; Fees for NMR and LC/MS. Structural and Biophysical Analysis: $119,500 1 Post-doctoral fellow, 1 FTE for 2 years; Chromatography media: Protein Concentrators; DNA for crystallization; Robot 96 well intelliplates; Synchroton data collection; Chemical. Tools for pharmacological studies : $12,000 Development LC-MS method for in vitro studies: PAMPA; Caco-2; Microsomal stability; CYP inhibition; Plasma protein binding by equilibrium dialysis; PK study in rats. 11

12 Target indication Lead Indications Proof-of-Concept for Monotherapy: Stratified colorectal cancer patients with mutations in either KRAS, NRAS or BRAF (~50% of cohort) Incidence rate (/10 5 )= 47; mortality rate= 17.5 Stratified lung cancer patients with mutations in KRAS, BRAF or NF1 (~50% of cohort) Incidence rate (/10 5 )= 62; mortality rate= 52.5 Secondary Clinical Indication primary tumor cells from patients where radiotherapy is standard of care but where resistance or relapse represents an unmet clinical need (breast, colorectal, ovarian, prostate). 12

13 Unmet medical need I There is no treatment that targets all RAS-driven tumors. the acute dependence of RAS-driven tumor cells on efficient oxidative DNA damage repair should be exploited in future therapeutic strategies. The successful use of a PARP1 inhibitor for the treatment of tumour cells in which BRCA1 or BRCA2 is inactivated provides a paradigm for the therapeutic exploitation of cancer cell addiction to a specific DNA repair pathway. Many cancer cells exhibit increased resistance to radiotherapy, leading to treatment failure and rapid relapse. radiotherapy is standard-of-care for more than 50% of cancer patients. chemotherapeutic agents used for radiosensitization cause adverse side-effects to nonirradiated tissues. CUT domain inhibitors would cause no or little side-effects to non-irradiated tissues. Therefore, there is an acute need to: develop targeted therapy for cancers in which the RAS pathway is activated. improve radiotherapy efficacy while reducing side effects. 13

14 Competition The increased efficiency of base excision repair (BER) in some cancers is currently underestimated. It is generally assumed that cancer cells adapt to elevated ROS production by increasing expression of ROS scavengers. Our survey, although limited, indicates that increased BER efficiency is more prevalent. Auxiliary factors in BER do not exist in bacteria. The role and importance of BER auxiliary factors in mammalian cells is only emerging now. Their implication in cancer has not yet been fully recognized. We have initiated a pipeline for the identification of other BER auxiliary factors. CUT domains stimulate OOG1, the DNA glycosylase that recognizes oxidized purines. We have identified auxiliary factors for NTHL1, the DNA glycosylase that recognizes oxidized pyrimidines. These factors are also implicated in cancer. Competitors We have no competitor working on the DNA repair function of CUX1 or CUT domains. In a future publication, we will include results refuting an article from Nature Genetics showing that CUX1 knockdown stimulates tumor growth of KE-37 T-cell lymphoma. Multiple CUX1 shrnas hinder proliferation of KE-37 cells. Collaborators Structural Biology: Dr. BushanNagar, Biochemistry, McGill U. Patient-Derived Tumors: Dr. Morag Park, Dr. Peter Siegel, GCRC, McGill U. Radiation Therapy: Dr. Jan Seuntjens, Radiation Oncology, MUHC. Medicinal Chemistry: Dr. Anne Marinier, IRIC, U. Montreal. Mouse Models: Dr. David Dankort, Biology, McGill U. Immunohistochemistry: Dr. Marie-Christine Guiot, Montreal Neurological Institute. 14

15 Comparables/Exit I am not aware of any relevant early-stage commercialisation or company start-up deals targeting a DNA repair auxiliary factor. AND Cancers in which the RAS pathway is activated (KRAS, NRAS, HRAS, BRAF, NF1) are still treated with conventional chemotherapy. There is yet no targeted therapy for these cancers. 15